ang4  (Santa Cruz Biotechnology)

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Impaired intestinal cell proliferation parallels increased senescence after burn injury in aged mice

doi: 10.1152/ajpgi.00176.2025

Figure Lengend Snippet: Expression of ang4 in the (A) colon, (B) IECs, and (C) organoids and camp in the (D) colon, (E) IECs, and (F) organoids measured by RT-qPCR across all groups shown as relative expression normalized to gapdh . *p<0.05 from Young Sham, +p<0.05 from young groups by one-way ANOVA with Tukey’s post-hoc test. n=3–10 mice combined from two independent experiments.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes, ang4 (Mm03647554_g1), camp (Mm00438285_m1), cdk4 (Mm00677718_g1), cdkn2a (Mm00494449_m1), lgr5 (Mm00438890_m1), and sox9 (Mm00448840_m1), (Applied Biosystems) and TaqMan Universal PCR Mastermix (cat#4364340, Applied Biosystems) and run on a QuantStudio 3 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell reports

Article Title: Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

doi: 10.1016/j.celrep.2022.111416

Figure Lengend Snippet: Figure 6. B. subtilis provides protection against the pathogen S. typhimurium in mouse small intestinal organoids (A) qPCR analysis of the levels of antimicrobial peptides (Defa1, Defa5, Ang4, and PLA2G2A) and TFF3 in small intestinal organoids with or without B. subtilis stimulation; n = 6. (B) Different treatment methods used in each group (Ctrl, B.s, S.t, B.s-S.t, and (DLL4+B.s)-S.t). (C) The morphology of the organoids in different groups was observed with a light microscope at 12 h post-infection. Scale bar, 200 mm. (D) The relative number of disrupted organoids (red arrow) in the different groups at 12 h post-infection; n = 6. (E) Photo used to determine plate counts in the different groups (Ctrl, B.s, S.t, B.s-S.t, and (DLL4+B.s)-S.t) at 12 h post-infection. (F) Number of invasive Salmonella in the organoids at 12 h post-infection; n = 6. (G) ELISA of inflammatory cytokine (TNF-a and IL-1b) levels in culture medium at 12 h post-infection; n = 6. Data represent the mean ± SD of three independent experiments; comparisons performed with t tests (two groups) or ANOVA (multiple groups). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant mouse TFF3 protein Novoprotein Cat#: C661 Cell-Light EdU DNA cell proliferation kit Guangzhou RiboBio Cat#: C10310-2 Recombinant JAG-1 protein R&D Systems Cat#: MAB599 Recombinant Ang4 protein R&D Systems Cat#: 964-AN-025 lipoteichoic acid (LTA) from Bacillus subtilis Sigma-Aldrich Cat#: L3265 FITC-UEA-1 Sigma-Aldrich Cat#: L9006 40,6-Diamidino-2-Phenylindole (DAPI) Invitrogen Cat#: D1306 IntestiCultTM Organoid Growth Medium (Mouse) STEMCELL Cat#: 06000 Fetal bovine serum VivaCell Cat#: C04001 Recombinant mouse Noggin protein PeproTech Cat#: 250-38 Recombinant mouse R-spondin1 protein PeproTech Cat#: 315-32 Recombinant Rat EGF protein T&L Biological Technology Cat#: GMP-TL676 Experimental models: Cell lines Human epithelial colorectal adenocarcinoma Chuanqiu Biotechnology Cat#: H010 Experimental models: Organisms/strains mouse: C57BL/6J Yangzhou University Animal Research Centre mouse: Smoc-Tlr2em1Smoc: C57BL/6J Nanjing University Nanjing Biomedical Research Institute mouse: Lgr5tm1(cre/ERT2)Cle: C57BL/6J (Hou et al., 2020a) Available from the Hou lab Oligonucleotides RT-qPCR primers This paper Table S1 Software and algorithms FlowJo Tree Star https://www.flowjo.com Prism Graphpad https://www.graphpad.com/scientific-software/prism/ ImageJ National Institutes of Health https://imagej.nih.gov/ij/

Techniques: Light Microscopy, Infection, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

doi: 10.1016/j.celrep.2022.111416

Figure Lengend Snippet: Figure 7. B. subtilis provides protection against the pathogen S. typhimurium in mice Mice were orally administered B. subtilis 168 (1 3 108 CFUs) or PBS as a control once a day for 21 days. Some mice were orally administered S. typhimurium strain 1344 (1 3 108 CFUs) once on day 14. (A) Changes in body weight were monitored daily starting from day 1 to day 19 and presented relative to the initial body weight; n = 6. (B) Time course of mouse survival; n = 6. (C) Representative pictures of mouse intestines. Graph showing small intestine and colon lengths; n = 6 mice. (D) Representative pictures of H&E staining in the ileum. Scale bar, 100 mm. Graph showing villus height and crypt depth; n = 6. (E) ELISA of antimicrobial peptides (Defa1, Defa5, Ang4, and PLA2G2A) and TFF3 expression in the ileum; n = 6. (F–H) ELISA of LPS in the serum and IL-1b and TNF-a in the ileum; n = 6. (I) Salmonella burden in the feces, as determined on streptomycin agar plates at 7 days post-infection. Figure shows the CFU per gram of fecal sample; n = 6. (J) Representative images of PAS staining in the ileum at day 21. Scale bar, 50 mm. Graph showing PAS+ cells per villus; n = 6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant mouse TFF3 protein Novoprotein Cat#: C661 Cell-Light EdU DNA cell proliferation kit Guangzhou RiboBio Cat#: C10310-2 Recombinant JAG-1 protein R&D Systems Cat#: MAB599 Recombinant Ang4 protein R&D Systems Cat#: 964-AN-025 lipoteichoic acid (LTA) from Bacillus subtilis Sigma-Aldrich Cat#: L3265 FITC-UEA-1 Sigma-Aldrich Cat#: L9006 40,6-Diamidino-2-Phenylindole (DAPI) Invitrogen Cat#: D1306 IntestiCultTM Organoid Growth Medium (Mouse) STEMCELL Cat#: 06000 Fetal bovine serum VivaCell Cat#: C04001 Recombinant mouse Noggin protein PeproTech Cat#: 250-38 Recombinant mouse R-spondin1 protein PeproTech Cat#: 315-32 Recombinant Rat EGF protein T&L Biological Technology Cat#: GMP-TL676 Experimental models: Cell lines Human epithelial colorectal adenocarcinoma Chuanqiu Biotechnology Cat#: H010 Experimental models: Organisms/strains mouse: C57BL/6J Yangzhou University Animal Research Centre mouse: Smoc-Tlr2em1Smoc: C57BL/6J Nanjing University Nanjing Biomedical Research Institute mouse: Lgr5tm1(cre/ERT2)Cle: C57BL/6J (Hou et al., 2020a) Available from the Hou lab Oligonucleotides RT-qPCR primers This paper Table S1 Software and algorithms FlowJo Tree Star https://www.flowjo.com Prism Graphpad https://www.graphpad.com/scientific-software/prism/ ImageJ National Institutes of Health https://imagej.nih.gov/ij/

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Infection

Journal: Gut Microbes

Article Title: Phospholipid metabolites of the gut microbiota promote hypoxia-induced intestinal injury via CD1d-dependent γδ T cells

doi: 10.1080/19490976.2022.2096994

Figure Lengend Snippet: Hypoxia Contributes to the Alteration of Gut Microbiota and Associated with Antimicrobial Peptides. (a-d) 16S rRNA analysis was performed on the cecal contents from SPF mice under hypoxia (5.0% oxygen concentration) or normoxia (20.9% oxygen concentration) for 48 hours (n = 6). (a) The relative abundance of intestinal microbiota at the phylum level and genus level. (b) Principal component analysis (PCA) and Principal co-ordinates analysis (PCoA) analysis based on the relative abundance of operational taxonomic units (97% similarity). (c) Volcano plot of differentially expressed genera. (d) The relative abundance of C. XIVa and Desulfovibrio in the cecal contents of normoxic and hypoxic mice was analyzed by 16S rRNA gene sequencing. (e) The relative abundance of C. XIVa and Desulfovibrio in the cecal contents of normoxic and hypoxic mice was quantified by Real-time qPCR analysis (n = 6). (f) Real-time qPCR analysis of Desulfovibrio in SPF mice after C. bolteae administered orally for 7 days (n = 6). (g) Real-time qPCR analysis of antimicrobial peptide Pla2, Ang4, Reg3g and Reg3b gene expression in the intestinal epithelium tissues (n = 6). (h) Correlation analysis between the relative content of C. XIVa and the relative expression of Ang4 or Pla2 in the intestine of SPF mice (n = 8). (i) Spectrophotometry of C. bolteae growth at different time points following recombinant Ang4 (5 µg/ml each) stimulation (n = 3). The data are representative of three independent experiments and shown by the mean ± SEM. *p <0.05, **p <0.01 by unpaired Student’s t -test (d, e, g, i), paired Student’s t -test (f) or linear regression analysis (h). ns, no significance.

Article Snippet: Recombinant Ang4 (Novoprotein, China) (5 μg/ml) was added to Reinforced Clostridium Medium (RCM) containing Clostridium bolteae (JCM 12243) and incubated at 37°C under anaerobic conditions for 6, 12, 24, 48 and 72 hours.

Techniques: Concentration Assay, Sequencing, Gene Expression, Expressing, Spectrophotometry, Recombinant

Journal: Gut Microbes

Article Title: Phospholipid metabolites of the gut microbiota promote hypoxia-induced intestinal injury via CD1d-dependent γδ T cells

doi: 10.1080/19490976.2022.2096994

Figure Lengend Snippet: Primer sequences.

Article Snippet: Recombinant Ang4 (Novoprotein, China) (5 μg/ml) was added to Reinforced Clostridium Medium (RCM) containing Clostridium bolteae (JCM 12243) and incubated at 37°C under anaerobic conditions for 6, 12, 24, 48 and 72 hours.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Structural overview and generation of WT and Mutant Angiogenin 4. (A) 3D structure of Ang4 (PDB entry 2J4T). The mutated residues in this study are highlighted in color and labeled. (B) Amino acid sequence of Ang4. (C) SDS-PAGE of purified WT Ang4 and its mutants. Proteins were electrophoresed on a 15% polyacrylamide gel and stained with the Coomassie Brilliant Blue R-250 dye.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Mutagenesis, Labeling, Sequencing, SDS Page, Purification, Staining

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Ang4 disrupts the membrane integrity of Salmonella typhimurium LT2. (A) Growth curve of bacteria treated with Ang4 measured by OD 600. (B) MIC and IC 50 of WT Ang4 determined by microbroth dilution method. (C) Killing percentage of Ang4-treated bacteria after 18 h incubation at 37°C. (D) Outer membrane permeabilization by Ang4 was evaluated by measuring the fluorescence intensity of NPN. (E,F) Cell membrane alteration in Ang4 treated bacteria was assessed by PI staining using flow cytometry. All data are shown as mean ± SD from three independent experiments. P -values were determined by two-tailed unpaired t -test or one-way ANOVA; *** P < 0.001 vs. control.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Membrane, Bacteria, Incubation, Fluorescence, Staining, Flow Cytometry, Two Tailed Test, Control

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Antimicrobial activity of Ang4 against Salmonella typhimurium LT2 is independent of RNase activity. (A) RNase activity of Ang4 and H12A determined by measuring the absorbance of perchloric acid soluble RNA fragments after 2 h incubation of assay mixtures at 37°C. (B) Growth profile of bacteria incubated with or without Ang4 or H12A at 37°C for 2 h. (C) Killing percentage of bacteria that were treated with PBS alone, Ang4, or H12A for 2 h before plating on LB agar plates. (D) Binding of Ang4 and H12A to bacteria was evaluated by ELISA. (E) The permeability of the bacterial outer membrane induced by Ang4 or H12A was assessed by measuring the fluorescence of NPN. (F,G) Flow cytometry to detect the inner membrane permeability of bacteria exposed to Ang4 or H12A using PI. All data are shown as mean ± SD from three independent experiments. P-values were determined by two-tailed unpaired t -test or one-way ANOVA with Tukey’s multiple comparisons test; ns > 0.05, *** P < 0.001 vs. WT Ang4.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Activity Assay, Incubation, Bacteria, Binding Assay, Enzyme-linked Immunosorbent Assay, Permeability, Membrane, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Cationic amino acids at the N-terminal α-helix are not responsible for binding but play a role in killing of bacteria. (A) Binding of Ang4 and its mutants to the bacterial cell membrane, as measured by ELISA at 450 nm. PBS was used as a control. (B) Salmonella growth curve after 2 h of incubation with or without Ang4 or its mutants. (C) Bacterial killing assay after 2 h of incubation with Ang4 or its mutants, as measured by colony count. (D) NPN uptake assay to identify bacterial outer membrane rupture following addition of Ang4 or its mutants. (E,F) PI staining to assess changes in bacterial cell membrane integrity after 2 h of treatment with Ang4 or its mutants. All data are presented as mean ± SD from three independent experiments. P-values were determined by one-way ANOVA with Tukey’s multiple comparisons test; ns > 0.05, ** P < 0.01, *** P < 0.001 vs. WT Ang4.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Binding Assay, Bacteria, Membrane, Enzyme-linked Immunosorbent Assay, Control, Incubation, Staining

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Cationic residues in the cell binding segment of Ang4 are crucial for the bacterial cell binding and killing. (A) RNase activity of Ang4 and its mutants was measured by absorbance at 260 nm. (B) Growth curve of bacteria after incubation with or without Ang4 or its mutants for 2 h. (C) Killing assay of bacteria performed 2 h post-incubation with Ang4 or its mutants estimated by colony count. PBS was used as a control. (D) Binding of Ang4 and its mutants to the bacterial cell membrane was estimated by ELISA at 450 nm. (E) NPN uptake assay to detect outer membrane disruption of bacteria after the addition of Ang4 or its mutants evaluated by increased fluorescence intensity. (F,G) PI staining to evaluate the alteration of bacterial cell membrane after 2 h post incubation at 37°C with Ang4 or its mutants. All data are presented as the mean ± SD from three independent experiments. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test; ns > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT Ang4.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Binding Assay, Activity Assay, Bacteria, Incubation, Control, Membrane, Enzyme-linked Immunosorbent Assay, Disruption, Fluorescence, Staining

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Overall structure of the gut microbiota community after administration of Ang4. (A) Observed features (ASVs) determined by Illumina MiSeq platforms in PBS- and Ang4-treated groups. (B) Rarefaction curves were calculated at multiple sequence depths. (C) Shannon entropy to compare differences in alpha diversity between the two groups. (D) Beta-diversity of the gut microbiota using unweighted UniFrac measures. P -values were determined using two-tailed unpaired t -test or one-way ANOVA with Tukey’s multiple comparisons test; ns > 0.05, ** P < 0.01.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Sequencing, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

doi: 10.3389/fmicb.2022.900948

Figure Lengend Snippet: Administration of Ang4 alters composition of gut microbiota. (A) Relative abundance of bacteria at the family level. (B) Relative abundance of the genera. All figures represent the fecal bacteria that were significantly altered between Ang4- and PBS-treated mice. P -values were determined by two-tailed unpaired t -test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: MIC and IC 50 of Ang4 protein were determined by GraphPad prism 9 using non-linear regression (curve fit).

Techniques: Bacteria, Two Tailed Test